System and method for solution phase gap peptide synthesis

ABSTRACT

Disclosed is a system and method for Fmoc/tBu solution-phase peptide synthesis including the development of a new benzyl-type GAP protecting group, and related uses thereto. This novel GAP protecting group is utilized in place of a polymer support, facilitating C to N Fmoc peptide synthesis without chromatography, recrystallization, or polymer supports. The GAP group can be added and removed in high yield.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Appl. Ser. No.62/270,432, filed Dec. 21, 2015, entitled “System And Method ForSolution Phase GAP Peptide Synthesis.” The foregoing patent applicationis hereby incorporated herein by reference in its entirety for allpurposes.

This application includes material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent disclosure, as it appears in thePatent and Trademark Office files or records, but otherwise reserves allcopyright rights whatsoever.

TECHNICAL FIELD

The present disclosure relates in general to the field of peptidesynthesis. In particular, the system provides for solution-phase peptidesynthesis without chromatography, recrystallization, or polymersupports, and allows for high overall yield and purity. The disclosedsystems and methods support a wide variety of scenarios and includevarious products and services.

STATEMENT OF FEDERALLY FUNDED RESEARCH

None.

BACKGROUND OF THE DISCLOSURE

Recent research efforts have made significant advancements m the area ofpurification chemistry, focusing specifically on avoiding columnchromatography and recrystallization. This research has been defined asGroup-Assisted Purification (GAP) chemistry/technology as a chemistryfor organic synthesis that avoids traditional purification methods suchas chromatography and/or recrystallization by purposefully introducing awell-functionalized group in the starting material or in the newlygenerated product. Such research has the potential to encompass theentire field of synthetic organic chemistry.

One area where protecting groups are used extensively is in peptidesynthesis, both for solid and solution phase approaches. Developed byMerrifield in the 1960's, Solid-Phase Peptide Synthesis (SPPS) hasbecome a standard protocol used by multiple scientific disciplines forresearch and manufacturing (See FIG. 1A). The advantages of the polymersupport lie in its ability to allow facile purification of the growingpeptide after each coupling/deprotection step, which avoids the use ofcolumn chromatography. The key disadvantage of SPPS lies in thedifficulty of scale-up: many polymer supports are expensive, and occupythe vast majority of the mass of the material to be worked with.Protecting groups are found in almost every complex synthesis wheremultiple functional groups are present. Effective protecting groups needto be robust to a wide variety of conditions, and must be added andremoved with high yield. An ideal example for GAP chemistry would be onein which a semi-permanent protecting group introduced the necessarysolubility characteristics required for GAP. However, most traditionalprotecting groups are nonpolar, and therefore do not generate therequired GAP solubility for most substrates. If a protecting group couldbe developed that generated adequate solubility control, then GAPchemistry could potentially be extended to all syntheses, which requirethe use of that protecting group.

Several approaches have been utilized. Published patent application WO2014093723 A2, teaches the protection of imines with a GAP-equippedchiral auxiliary, then using these chiral, N-phosphonyl imines aselectrophiles in asymmetric boron addition reactions. Purification wasconducted via GAP processes. This work is valuable in that it providesfacile access to chiral, α-boronic acid amines, which could potentiallybe used to synthesize novel amino acid derivatives, which couldpotentially be incorporated into novel peptide targets.

U.S. Pat. No. 8,383,770 B2 teaches the use of the Fmoc and BocN-terminus protecting groups in SPPS. This technology is well known andwidely applied in industry. Boc and Fmoc groups have been used fordecades in all areas of peptide chemistry, and the preferred Fmoc groupis almost entirely restricted to solid phase. Examples of economicallyfeasible Fmoc protection schemes in solution are non-existent, with fewexamples in the literature at all.

U.S. Pat. No. 5,516,891 A provides one of the few examples of Fmoc-basedSolPPS. Again, the Fmoc peptide synthesis is almost entirely restrictedto SPPS, due to the formation of N-fluorenylmethylpiperidine (NFMP) as aside product during deprotection, which is difficult to remove withoutpolymer supports. The standard protocol for Fmoc deprotection is to stirthe Fmoc-peptide in a solution of DMF or DCM with excess piperidine,deprotecting the Fmoc group and forming NFMP in the process. The '891patent teaches removal of this impurity by deprotecting with4-aminomethylpiperidine (4AMP) instead of piperidine. This formsNFMP-CH2NH2 instead of NFMP, which due to the presence of the extraamino group, can be extracted into water. The problem with this methodlies in the high cost of using 4AMP. Per Sigma Aldrich, 4AMP costs $3.80per gram, while piperidine only costs $0.12 per gram. This is why thismethod is cost prohibitive, and why it has not been accepted by theindustry.

It is therefore a need in the art to develop an economically feasibleGAP peptide synthesis system capable of overcoming these limitations,while keeping the purification benefits of solid phase peptidesynthesis.

SUMMARY OF THE DISCLOSURE

The present disclosure addresses failings in the art by providing asystem and method for peptide synthesis utilizing a reaction whichoccurs in solution phase, without the mass waste of polymer supports,but retains all of the purification benefits of SPPS as an alternativeto both traditional solution-phase peptide synthesis (SolPPS) as well asSPPS, affording advantages of both methods. By utilizing the advantagesof GAP chemistry, an Fmoc-SolPPS strategy is presented that iseconomically feasible and useful for the commercial production ofpeptides.

It is therefore an object of the present disclosure to enable GAPpeptide synthesis (GAP-PS) via the development of a new GAP benzyl-typeprotecting group for C-terminus protection (See FIG. 1B). In connectionwith C-terminus protection, GAP-PS may be achieved using an Fmoc/tBustrategy, which is the most used method in SPPS due to its milddeprotection protocols. This strategy is currently almost entirelyrestricted to SPPS due to the formation of N-fluorenylmethylpiperidine(NFMP) as a side product during deprotection, which is difficult toremove without polymer supports. It is therefore an object of thepresent disclosure to provide over 1 gram of target peptide, such asthymopentin, in high yield and high purity via utilization of asolution-phase Fmoc/tBu strategy as an example for a general method ofpeptide synthesis. Protection of various amino acids with this newprotecting group has also been achieved in consistent quantitativeyield.

In one aspect, a method for peptide synthesis is provided. The methodallows for a high yield (over 50%) with high purity (99%) using theFmoc/tBut strategy with solution-phase peptide synthesis (SolPPS). Thepresent invention utilizes Group-Assisted Purification (GAP) inconjunction with SolPPS, enabling the peptide to be purified throughprecipitation instead of recrystallization or chromatography. Thedisclosed method also avoids solid-phase peptide synthesis (SPPS),thereby increasing the amount of product that is actually formed

It is another object of the present invention to provide a novelC-terminus protecting group (referred to herein as “BnDppOH”, “BnDppYH”,“BzDppOH”) which is chemically linked to the C-terminus. The use of thisGAP group is also different: whereas previous GAP groups served as aminoprotecting groups, the present invention discloses a protecting groupfor the carboxylic acid. By protecting the carboxylic acid, peptidesynthesis is allowed in an industry-preferred C to N direction ratherthan the N to C direction, a critical difference from previous GAP-PSmethods, further enabling the use of Fmoc as a temporary protectinggroup with which to grow the peptide chain. During Fmoc deprotection,NFMP is formed which is difficult to remove without solid supports. Thepresent invention provides a method of removal to selectivelyprecipitate the GAP-peptide, thereby leaving NFNIP in solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of thedisclosure will be apparent from the following description ofembodiments as illustrated in the accompanying drawings, in whichreference characters refer to the same parts throughout the variousviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating principles of the disclosure:

FIG. 1A depicts a prior art process of Solid Phase Peptide Synthesis(SPPS).

FIG. 1B depicts a process of the present disclosure including the use ofa benzyl-type protecting group for C-terminus protection.

FIG. 2 depicts a process for development of a protecting group utilizedin FIG. 1B.

FIG. 3 depicts a schematic for testing the orthogonality and GAPcapability of the protecting group of FIG. 2.

FIGS. 4A-4B each depicts a schematic for the process of attaching theprotecting group of FIG. 2 to various amino acids.

FIG. 5 depicts a schematic for the synthesis of thymopentin using theprotecting group of FIG. 2 for purposes of the exemplary non-limitingexample of peptide synthesis of the present invention.

FIG. 6 depicts other protecting groups that can be use in embodiments ofthe present invention.

FIG. 7 depicts an alternative process for production, synthesis andmanufacture of the protecting groups of FIG. 6 as utilized inembodiments of the present invention.

FIG. 8 depicts another alternative process for production, synthesis andmanufacture of protecting groups of FIG. 6 as utilized in embodiments ofthe present invention.

FIG. 9A depicts a schematic for the process of attaching the protectinggroup of “BnDppYH” to various amino acids.

FIG. 9B depicts the protecting group “BnDppYH” utilized in the schematicfor the process shown in FIG. 9A.

FIG. 10A depicts a schematic for the process of attaching the protectinggroup of “BnDppZH” to various amino acids.

FIG. 10B depicts the protecting group “BzDppOH” utilized in theschematic for the process shown in FIG. 10A.

DETAILED DESCRIPTION OF THE DISCLOSURE

While the making and using of various embodiments of the presentdisclosure are discussed in detail below, it should be appreciated thatthe present disclosure provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts, goods, orservices. The specific embodiments discussed herein are merelyillustrative of specific ways to make and use the disclosure and do notdelimit the scope of the disclosure.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this disclosure pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The present disclosure will now be described more fully hereinafter withreference to the accompanying drawings, which form a part hereof, andwhich show, by way of illustration, specific example embodiments.Subject matter may, however, be embodied in a variety of different formsand, therefore, covered or claimed subject matter is intended to beconstrued as not being limited to any example embodiments set forthherein; example embodiments are provided merely to be illustrative.Likewise, a reasonably broad scope for claimed or covered subject matteris intended. Among other things, for example, subject matter may beembodied as methods, compositions, or systems. Accordingly, embodimentsmay, for example, take the form of methods, compositions, compounds,materials, or any combination thereof. The following detaileddescription is, therefore, not intended to be taken in a limiting sense.

Throughout the specification and claims, terms may have nuanced meaningssuggested or implied in context beyond an explicitly stated meaning.Likewise, the phrase “in one embodiment” as used herein does notnecessarily refer to the same embodiment and the phrase “in anotherembodiment” as used herein does not necessarily refer to a differentembodiment. It is intended, for example, that claimed subject matterinclude combinations of example embodiments in whole or in part.

In general, terminology may be understood at least in part from usage incontext. For example, terms, such as “and”, “or”, or “and/or,” as usedherein may include a variety of meanings that may depend at least inpart upon the context in which such terms are used. Typically, “or” ifused to associate a list, such as A, B or C, is intended to mean A, B,and C, here used in the inclusive sense, as well as A, B or C, here usedin the exclusive sense. In addition, the term “one or more” as usedherein, depending at least in part upon context, may be used to describeany feature, structure, or characteristic in a singular sense or may beused to describe combinations of features, structures or characteristicsin a plural sense. Similarly, terms, such as “a,” “an,” or “the,” again,may be understood to convey a singular usage or to convey a pluralusage, depending at least in part upon context. In addition, the term“based on” may be understood as not necessarily intended to convey anexclusive set of factors and may, instead, allow for existence ofadditional factors not necessarily expressly described, again, dependingat least in part on context.

It is therefore an embodiment of the present disclosure to provide asystem and method for a new C-terminus protecting group. In designingthe new protecting group, it was apparent that the GAP-functionalizedsegment of the protecting group would need to be stable to a widevariety of conditions. Considerations were taken that it must providethe necessary solubility characteristics for GAP chemistry. Also, itmust work efficiently and orthogonally with the reactivity of currentprotection strategies. A modified benzyl protecting group was thusutilized in order to keep the desirable reactivity while introducing theGAP group. The GAP group chosen is diphenylphosphine oxide, due to knownprevious success with phosphine oxide groups using GAP chemistry. Also,attachment of this group onto the para position of the benzyl groupcreates a triphenylphosphine oxide moiety, which is widely known in theliterature to be stable to an extensive variety of conditions. Thisstability is necessary to avoid interference with the multipledeprotection conditions that the substrate may be exposed to, therebyestablishing true orthogonality.

Initial efforts focused on the development of chiral, N-phosphonyl andN-phosphinyl imine chemistry for the synthesis of chiral amines, withmuch success. By controlling solubility, the chiral amine products canbe selectively precipitated from the crude mixture, thereby avoidingchromatography and recrystallization. Further efforts have extended thistechnology to other substrates and functional groups. In order to dothis, the GAP properties are taken from chiral auxiliaries and, withmodification, present the basis for the GAP protecting groups of thepresent disclosure.

In an exemplary embodiment of the present disclosure, synthesis of thisnew protecting group begins with commercially available,diphenyl(p-tolyl)phosphine 1 (FIG. 2). Oxidation of 1 with potassiumpermanganate provides benzoic acid 2, as well as the GAP group throughphosphine oxidation. This GAP group is diphenylphosphine oxide (Dpp).Esterification followed by borohydride reduction affords theGAP-equipped benzyl alcohol 4, or “BndppOH” (alternatively “HOBndpp”),in high yield. Next, the orthogonality and the GAP capabilities of thisnew protecting group are tested. Protection of Boc-Phe-OH was bothfacile and quantitative using EDCI as the carbodiimide coupling reagent(FIG. 3). The product 5a can be selectively precipitated from an ethylacetate/petroleum ether solvent mixture as a white solid, thereby

satisfying the requirements of GAP chemistry. Deprotection of the Bocgroup was also quantitative, and did not result in any loss of the Bndppgroup. The Bndpp group can be easily removed using catalytichydrogenation, and also can be recovered and recycled as “HBndpp” 7a forreuse after washing away the deprotected amino acid. Subjection of 7a topermanganate oxidation affords 2, which can be transformed into HOBndpp4 as previously mentioned (FIG. 2).

FIGS. 4A-4B depict a schematic for the process of attaching theprotecting group of FIG. 2 to various amino acids. A full substratescope for amino acid protection is shown below in TABLE 1, withconsistent quantitative yields for the protection of a variety of Bocand Fmoc amino acids with varying side-chain protecting groups. Of noteis the quantitative protection of tryptophan, arginine, valine, andcysteine.

TABLE 1 Product PG- -AA- Yield 5a Boc- -Phe- 99% 5b Boc- -Cys(Acm)- 99%5c Fmoc- -Lys(Boc)- 99% 5d Fmoc- -Asp(tBu)- 99% 5e Fmoc- -Trp(Boc)- 97%5f Fmoc- -Arg(Pbf)- 99% 5g Fmoc- -Val- 99% 5h Fmoc- -Asn(Trt)- 99% 5iFmoc- -Ala- 99% 5j Fmoc- -Gly- 99% 5k Fmoc- -Tyr(tBu)- 99%

In one embodiment a method for Fmoc/tBu liquid-phase peptide synthesisvia GAP chemistry/technology is presented, along with the development ofa new benzyl-type GAP protecting group for carboxylic acids. This newGAP protecting group is utilized in place of a polymer support, andfacilitates C to N Fmoc peptide synthesis without chromatography orrecrystallization. The GAP protecting group can be added and removed inhigh yield, while maintaining an orthogonal relationship to the otherprotecting groups present. As a first test of this new protecting groupfor GAP peptide synthesis, over 1 gram of the pentapeptide drugthymopentin (an immunostimulant) was synthesized in high overall yield(83%) and high purity (99%).

In one embodiment of the present invention, a protecting group ispresented for Group Assisted Purification (GAP) peptide synthesis,comprising the following compounds:

wherein: R is: H, Me, or OMe; Y is: O, S, and NH; and X is: O, S, or NH.

In another embodiment, the present invention provides a method offorming a protecting group for C-terminus protection, comprising ofFmoc-tBu-based solution phase peptide synthesis (SolPPS). The methodincludes protecting group:

which is produced by the following:

wherein, said protecting group IS formed by refluxing(p-tolyl)diphenylphosphine with potassium permanganate (KMnO4),isolating the carboxylic acid product, refluxing the carboxylic acidproduct in acidic ethanol (EtOH, H+), and adding sodium borohydride(NaBH4).

In another embodiment, the present invention presents a method ofproducing protecting group:

produced by the following:

wherein the protecting group is produced by reacting dibromide withbutyllithium (nBuLi), diphenylchlorophosphine (Ph2PC1), carbon dioxide(CO2), and hydrogen peroxide (H+, H202) in a single reaction to producea carboxylic acid product, isolating the carboxylic acid product,refluxing the carboxylic acid product in acidic ethanol (EtOH, H+),adding sodium borohydride (NaBH4) to form the alcohol product, andtreating the alcohol product with mesic anhydride Ms2O or YH2; andwherein R is: H, Me, or OMe; and Y is: S or NH.

In another embodiment, protecting group:

is produced by the following:

wherein the ethyl ester derivative is reacted withdiphenylchlorophosphine (Ph2PC1), followed by hydrogen peroxide (H₂O₂)for oxidation. The resulting phosphine oxide is treated with sodiumborohydride (NaBH4) to produce the alcohol, which is treated with mesicanhydride (Ms2O) and YH2 to form (1C).; and wherein R is: H, Me, or OMe;X is: O, or NH; and Y is: O, S, or NH.

In another embodiment protecting group:

is produced by the following:

wherein the dibromide is reacted with butyllithium (nBuLi),diphenylchlorophosphine (Ph2PC1), carbon dioxide (CO2), and hydrogenperoxide (H+, H202) in a one-pot fashion to produce the carboxylic acidproduct (ID).; and wherein R is: H, Me, or OMe.

In another embodiment of the present invention, protecting group:

is produced by the following:

wherein the ethyl ester derivative is reacted withdiphenylchlorophosphine (Ph2PC1), followed by hydrogen peroxide (H2O2)for oxidation. The resulting phosphine oxide is treated with lithiumhydroxide (LiOH) and water (H2O), forming the carboxylic acid product(IE).; and wherein R is: H, Me, or OMe; and X is: O, S or NH.

In another embodiment of the present invention, a method of attaching aprotecting group 1A, 1B, 1C, 1D, or 1E to an amino acid, wherein themethod comprises reacting a protecting group of claim 1 with amino acidcompound:

Such method may comprise the steps of:

wherein BnDppOH is a protecting group (1A, 1B, 1C, 1D, or 1E); andwherein Pg may include, but is not limited to: Cbz, Fmoc, Boc, Bn, Fm,or tBu; and wherein Z is a general variable.

In another embodiment, a method of the present invention includes theprotecting group:

The method may further comprise the steps of:

wherein BnDppYH is a protecting group (1A, 1B, 1C, 1D, or 1E); andwherein Pg may include but is not limited to: Cbz, Fmoc, Boc, Bn, Fm, ortBu; and wherein Z is a general variable.

In another embodiment of the present invention the method includes aprotecting group:

wherein R is: H, Me, or OMe; and wherein X is: O, S, or NH.

In another embodiment the method comprises the steps of:

wherein BnDppYH is a protecting group 1A, 1B, 1C, 1D, or 1E; Pg mayinclude but is not limited to: Cbz, Fmoc, Boc, Bn, Fm, or tBu; andwherein Z is a general variable.

In another embodiment, the method of the present invention comprises thesteps of:

wherein BzDppOH is a protecting group 1A, 1B, 1C, 1D, or 1E; Pg mayinclude but is not limited to: Cbz, Fmoc, Boc, Bn, Fm, or tBu; and Z isa general variable.

In another embodiment, the method comprises the protecting group:

wherein R is selected from the group consisting of: H, Me, or OMe; andwherein X is selected from the group consisting of: O, S, or NH.

In another embodiment of the present invention, a method of performing aGroup Assisted Purification (GAP) peptide synthesis is provided, whereinthe method comprises the steps of attaching a protecting group 1A, 1B,1C, 1D, or 1E to an amino acid using any of the methods described hereinand then Fmoc-tBu-based solution phase peptide synthesis (SolPPS)coupling reactions on the resulting products the methods describedherein. Such method of GAP-PS may further include the reaction occurringin ethyl acetate, or alternatively in dichloromethane.

The principles discussed herein may be embodied in many different forms.The preferred embodiments of the present disclosure will now bedescribed where for completeness, reference should be made at least tothe Figures.

Example 1

For a first application of a new protecting group, capabilities inhandling an Fmoc/tBu SolPPS strategy are tested. The target peptide ofinterest for this non-limiting example is thymopentin, apharmacologically interesting, biologically active pentapeptide subunitof the immunomodulatory polypeptide, thymopoietin. For a short peptide,thymopentin contains amino acids with a variety of functional groups (1aromatic, two basic (one with guanidine), two acidic, and oneB-branched). This makes thymopentin an ideal candidate for an exemplaryuse of a GAP protecting group and its ability to tolerate the removal ofseveral side-chain protecting groups. Synthesis of thymopentin isillustrated in FIG. 5. Compound 5k is first treated with 30% piperidinein DCM for 10 minutes to remove the Fmoc group, followed by ammoniumchloride wash to remove the excess piperidine. The DCM layer (afterdrying) is directly loaded with the next Fmoc ammo acid (side chainprotection as noted), along with TBTU coupling reagent and DIPEA. Aftercoupling for 20 minutes, the reaction mixture is washed with ammoniumchloride and 0.5 M sodium hydroxide (respectively), dried and evacuated.The crude product after coupling contains several impurities, mostnotably NFMP and tetramethyl urea (from coupling). The GAP purificationprocedure can easily remove these impurities simply by dissolving themixture in a minimal amount of ethyl acetate, followed by selectiveprecipitation of the GAP-peptide with petroleum ether. For the tetra-and pentapeptide fragments, a small amount of DCM is added to the ethylacetate prior to precipitation, to help with the solubility. Followingthe last coupling step and the synthesis of 9k, the last Fmoc group isremoved as before but after workup, the DCM layer is concentrated andthe peptide is dissolved in TFA/DCM/H₂O (6/3/1) solution for side-chaindeprotection. The pentapeptide 10k (now with Bndpp as the onlyprotecting group) is precipitated using diethyl ether. This peptide isthen subjected to hydrogenation and the GAP group removed. The productis isolated via extraction from chloroform with 10% acetic acid (aq).The product is isolated via extraction from chloroform with 10% aceticacid (aq). Unexpectedly, HPLC analysis of the product peptide revealsthat the compound is nearly 99% pure without any column chromatography,recrystallization, or polymer supports. The GAP group can be recoveredsimply by evacuating the chloroform layer after extraction. Subjectingthis raw material to the synthesis methods in FIG. 2 can regenerateBndppOH

General Methods:

All solvents were ACS grade and used without additional purification.FIRMS analysis was performed using an Orbitrap mass analyzer. HPLCanalysis was conducted using a Perkin Elmer Flexar isocratic pumpequipped with a UV detector. Fmoc and Boc protected amino acids werepurchased from BachemBio and used directly for coupling.

Synthesis of Benzoic Acid 2:

10.0 g 1 was placed in a 500 mL round-bottomed flask, followed by 130 mL0.43 M NaOH(aq) solution and then 22.2 g KMn0₄. The reaction was stirredat reflux for 12 hours, after which the reaction mixture was filteredthrough celite while hot. The resulting solution was washed X2 withdiethyl ether, followed by the addition of 50% H₂ S0₄ to precipitate theproduct. After filtration, benzoic acid 2 was collected as a whitesolid; yield, 10.8 g, 93%; this product was directly subjected to thenext reaction.

Synthesis of Ester 3:

10.8 g 2 was placed in a 500 mL round-bottomed flask along with 300 mLethanol and 3 mL thionyl chloride. The reaction was brought to refluxand stirred for 12 hours. After completion, the reaction was cooled toroom temperature and the volatiles evacuated, affording ester 3 as awhite solid; yield, 11.8 g, 99%; this product was directly subjected tothe next reaction.

Synthesis of BndppOH 4:

11.8 g ester 3 was placed in a 500 mL round-bottomed flask along with300 mL ethanol. The reaction was cooled to 0° C., after which 3.82 gNaBH₄ was added portionwise. The reaction was brought to roomtemperature and stirred for 12 hours. The solvent was evacuated,followed by solvation of the crude in DCM and washing X3 with 2 MHCl(aq). The organic layer was then dried over MgSO₄, filtered, andevacuated to afford BndppOH 4 as a white solid; yield, 9.96 g, 96%; thiscompound has been previously synthesized via a different method, and NMRdata matches that found in the literature³⁰: ¹H NJVIR (400 MHz, CDCl₃)δ=7.62-7.57 (m, 4H), 7.54-7.47 (m, 4H), 7.45-7.40 (m, 4H), 7.38-7.36 (m,2H), 4.70 (s, 2H).

General Procedure for Bndpp Protection:

100 mg BndppOH, 2.0 eq PG-AA-OH, and 10 mL DCM were stirred at 0° C. ina 20 mL screw-cap vial. 124 mg (2.0 eq) EDCI(HCl) was added, and thereaction was stirred for 10 min, at which point 4 mg (10 mol %) DMAP wasadded and the reaction was brought to room temp and stirred for 2 hours.The reaction mixture was washed X2 with sat. NH₄Cl(aq), followed by sat.Na₂CO₃(aq) X2. The combined organic layers were dried with MgSO₄,filtered, and evacuated to afford the crude protected amino acid. GAPpurification was performed by dissolving the crude mixture in a minimalamount of ethyl acetate, followed by precipitation with petroleum etherand filtration of the resulting white precipitate. This same procedurewas used for every substrate except 5k, where the reaction was conductedon a larger scale using 600 mg BndppOH and the same equivalents of theother reagents as before.

Compound Legend

Compound 5a. White solid; yield 180 mg, 99%; mp 62-63° C.; ¹H NMR (400MHz, CDCl3) δ=7.69-7.63 (m, 6H), 7.58-7.52 (m, 2H), 7.49-7.45 (m, 4H),7.35-7.32 (m, 2H), 7.24-7.18 (m, 3H), 7.07-7.05 (d, J=6.4 Hz, 2H),5.20-5.12 (m, 2H), 4.96-4.95 (d, J=7.8 Hz, 1H), 4.68-4.58 (m, 1H),3.09-3.07 (d, J=5.9 Hz, 2H), 1.40 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ=171.9, 155.2, 139.3, 135.9, 133.0, 132.6, 132.5, 132.2, 132.1, 132.0,129.4, 128.8, 128.6, 128.2, 128.1, 127.2, 80.2, 66.3, 54.6, 38.5, 28.4;³¹P NMR (162 MHz, CDCl₃) δ=29.28; HRMS (ESI): m/z calcd for[C₃₃H₃₄NO₅P+H]⁺: 556.2253, found: 556.2235.

Compound 5b. White solid; yield 189 mg, 99%; mp 76-77° C.; ¹H NMR (400MHz, CDCl₃) δ=7.69-7.64 (m, 6H), 7.58-7.54 (m, 2H), 7.49-7.44 (m, 6H),6.65 (bs, 1H), 5.52-5.50 (d, J=5.9 Hz, 1H), 5.27-5.19 (m, 2H), 4.54 (bs,1H), 4.38-4.32 (m, 2H), 3.09-2.91 (m, 2H), 2.06-2.00 (m, 1H), 1.98 (s,3H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ=170.9, 170.4, 139.3,133.5, 132.8, 132.6, 132.5, 132.4, 132.2, 132.1, 131.8, 128.7, 128.2,80.7, 66.7, 54.2, 42.2, 34.5, 28.4, 23.3, 22.5, 14.2; ³¹P NMR (162 MHz,CDCl₃) δ=29.46; HRMS (ESI): m/z calcd for [C₃₀H₃₅N₂O₆PS+H]⁺: 583.2032,found: 583.2012.

Compound 5c. White solid; yield 246 mg, 99%; mp 86-87° C.; ¹H NMR (400MHz, CDCl₃) δ=7.76-7.74 (d, J=7.5 Hz, 2H), 7.69-7.63 (m, 6H), 7.60-7.52(m, 4H), 7.47-7.42 (m, 6H), 7.40-7.36 (t, J=7.4 Hz, 2H), 7.31-7.27 (t,J=7.4 Hz, 2H), 5.48-5.46 (d, J=7.3 Hz, 1H), 5.22 (s, 2H), 4.65-4.57 (bs,1H), 4.43-4.34 (m, 3H), 4.22-4.19 (t, J=6.9 Hz, 1H), 3.10-3.02 (m, 2H),1.88-1.84 (m, 1H), 1.72-1.68 (m, 1H), 1.42 (s, 9H), 1.38-1.24 (m, 4H);¹³C NMR (100 MHz, CDCl₃) δ=172.4, 156.2, 143.9, 141.4, 139.5, 132.9,132.6, 132.2, 131.8, 128.7, 128.0, 127.8, 127.2, 125.2, 120.1, 79.3,67.2, 66.4, 54.0, 47.3, 40.0, 32.1, 29.8, 28.5, 22.5; ³¹P NMR (162 MHz,CDCl₃) δ=29.37; HRMS (ESI): m/z calcd for [C₄₅H₄₇N₂O₇P+H]⁺: 759.3199,found: 759.3183.

Compound 5d. White solid; yield 227 mg, 99%; mp 85-86° C.; ¹H NMR (400MHz, CDCl₃) δ=7.76-7.74 (d, J=7.5 Hz, 2H), 7.66-7.52 (m, 10H), 7.46-7.36(m, 8H), 7.29-7.26 (t, J=7.2 Hz, 2H), 5.86-5.84 (d, J=8.6 Hz, 1H),5.29-5.20 (dd, J=12.8 Hz, 12.4 Hz, 2H), 4.69-4.66 (m, 1H), 4.44-4.31 (m,2H), 4.24-4.21 (t, J=7.0 Hz, 1H), 3.01-2.95 (dd, J=4.3 Hz, 17.0 Hz, 1H),2.81-2.76 (dd, J=4.2 Hz, 17.0 Hz, 1H), 1.39 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ=170.9, 170.2, 156.1, 143.9, 143.8, 141.4, 139.5, 132.7, 132.6,132.5, 132.2, 132.1, 131.7, 128.7, 128.6, 128.0, 127.9, 127.2, 125.2,120.1, 82.1, 67.4, 66.7, 50.7, 47.2, 37.8, 28.1; ³¹P NMR (162 MHz,CDCl₃) δ=29.75; HRMS (ESI): m/z calcd for [C₄₂H₄₀NO₇P+H]⁺: 702.2621,found: 702.2602.

Compound 5e. White solid; yield 257 mg, 97%; mp 98-99° C.; ¹H NMR (400MHz, CDCl₃) δ=8.10-8.08 (d, J=7.7 Hz, 1H), 7.76-7.74 (d, J=7.5 Hz, 2H),7.68-7.61 (m, 6H), 7.56-7.36 (m, 14H), 7.31-7.25 (m, 3H), 7.21-7.18 (t,J=7.5 Hz, 1H), 5.48-5.46 (d, J=8.2 Hz, 1H), 5.21-5.06 (dd, J=12.9, 47.8Hz, 2H), 4.84-4.79 (m, 1H), 4.41-4.34 (m, 2H), 4.22-4.18 (t, J=7.0 Hz,1H), 3.28-3.27 (d, J=5.7 Hz, 2H), 1.63 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ=171.6, 155.8, 149.6, 143.9, 143.8, 141.4, 139.1, 135.5, 132.9, 132.6,132.5, 132.2, 132.1, 131.8, 130.4, 128.7, 128.6, 127.8, 127.2, 125.2,124.8, 124.3, 122.8, 120.1, 118.9, 115.5, 114.8, 84.0, 67.4, 66.6, 54.3,47.2, 28.2; ³¹P NMR (162 MHz, CDCl₃) δ=29.32; HRMS (ESI): m/z calcd for[C₅₀H₄₅N₂O₇P+H]⁺: 817.3043, found: 817.3031.

Compound 5f. White solid; yield 304 mg, 99%; mp 117-118° C.; ¹H NMR (400MHz, CDCl₃) δ=7.75-7.73 (d, J=7.5 Hz, 2H), 7.69-7.63 (m, 4H), 7.60-7.55(m, 4H), 7.53-7.46 (m, 8H), 7.39-7.35 (t, J=7.4 Hz, 2H), 7.29-7.27 (d,J=7.4 Hz, 2H), 6.61 (bs, 2H), 5.88 (bs, 1H), 5.50-5.35 (dd, J, =9.7 Hz,J2=52.8 Hz, 2H), 5.03-5.00 (d, J=11.8 Hz, 1H), 4.36-4.34 (m, 3H),4.20-4.16 (t, J=7.0 Hz, 1H), 3.25-3.15 (m, 2H), 2.90 (s, 2H), 2.78-2.67(m, 2H), 2.58 (s, 3H), 2.51 (s, 3H), 2.06 (s, 3H), 1.68-1.57 (m, 2H),1.42 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ=172.0, 158.6, 156.6, 156.2,143.8, 141.3, 140.0, 138.3, 133.3, 132.5, 132.4, 132.2, 132.0, 131.9,131.8, 130.8, 128.9, 128.8, 127.8, 127.2, 125.2, 124.6, 121.1, 120.0,119.8, 117.4, 86.4, 68.0, 67.2, 66.2, 53.5, 47.1, 43.3, 40.5, 29.6,28.6, 25.2, 19.4, 18.1, 12.6; ³¹P NMR (162 MHz, CDCl₃) δ=31.03; HRMS(ESI): m/z calcd for [C₅₃H₅₅N₄O₈PS+H]⁺: 939.3556, found: 939.3538.

Compound 5g. White solid; yield 204 mg, 99% mp 81-82° C.; ¹NMR (400 MHz,CDCh) δ=7.77-7.75 (d, J=7.2 Hz, 2H), 7.70-7.53 (m, 10H), 7.48-7.44 (m,6H), 7.41-7.37 (t, J=7.2 Hz, 2H), 7.32-7.28 (t, J=7.2 Hz, 2H), 5.36-5.34(d, J=8.8 Hz, 1H), 5.22 (s, 2H), 4.44-4.32 (m, 3H), 4.24-4.21 (t, J=6.8Hz, 1H), 2.26-2.17 (m, 1H), 0.97-0.95 (d, J=6.8 Hz, 3H); ¹³C NMR (100MHz, CDCl₃) δ=172.0, 156.3, 143.9, 143.8, 141.4, 139.4, 132.6, 132.5,132.2, 132.1, 128.7, 128.6, 128.1, 128.0, 127.8, 127.1, 125.1, 120.1,67.1, 66.2, 59.1, 47.2, 31.3, 19.1, 17.6; ³¹P NMR (162 MHz, CDCl₃)8=29.45; HRMS (ESI): m/z calcd for [C₃₉H₃₆NO₅P+H]⁺: 630.2409, found:630.2392.

Compound 5h. White solid; yield 287 mg, 99%; mp 121-122° C.; ¹H NMR (400MHz, CDCl₃) δ=7.76-7.71 (t, J=6.4 Hz, 2H), 7.65-7.51 (m, 12H), 7.46-7.40(m, 4H), 7.38-7.31 (m, 4H), 7.24-7.20 (m, 9H), 7.15-7.13 (m, 6H), 6.75(s, 1H), 6.13-6.11 (d, J=8.8 Hz, 1H), 5.21-5.11 (q, J=12.8 Hz, 2H),4.69-4.65 (m, 1H), 4.43-4.38 (m, 1H), 4.30-4.26 (t, J=8.9 Hz, 1H),4.20-4.16 (t, J=7.1 Hz, 1H), 3.18-3.13 (dd, J₁=4.2 Hz, J₂=15.8 Hz, 1H),2.87-2.82 (dd, J₁=4.2 Hz, J₂=15.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃)δ=171.0, 169.4, 156.4, 144.3, 144.0, 143.8, 141.4, 139.6, 132.6, 132.5,132.2, 132.0, 128.7, 128.6, 128.2, 127.9, 127.6, 127.5, 127.4, 127.2,125.3, 120.1, 71.1, 67.4, 66.6, 51.2, 47.2, 38.8; ³¹P NMR (162 MHz,CDCl₃) δ=29.38; HRMS (ESI): m/z calcd for C₅H₄₇N₂O₆P+H]⁺: 887.3250,found: 887.3230.

Compound 5i. White solid; yield 195 mg, 99%; mp 78-79° C.; ¹H NMR (400MHz, CDCl₃) δ=7.76-7.75 (d, J=7.6 Hz, 2H), 7.70-7.63 (m, 6H), 7.59-7.53(m, 4H), 7.48-7.37 (m, 8H), 7.31-7.28 (t, J=7.6 Hz, 2H), 5.37-5.35 (d,J=7.6 Hz, 1H), 5.23 (s, 2H), 4.49-4.38 (m, 3H), 4.23-4.19 (t, J=7.2 Hz,1H), 1.46-1.44 (d, J=7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ=172.9,155.8, 144.0, 143.8, 141.4, 139.5, 132.9, 132.6, 132.5, 132.2, 132.1,131.9, 128.7, 128.6, 127.9 127.8, 127.2, 125.2, 120.1, 67.2, 66.4, 53.6,49.8, 47.3, 31.7, 22.8, 18.7, 14.3; 31P NMR (162 MHz, CDCl₃) δ=29.28;HRMS (ESI): m/z calcd for [C₃₇H₃₂NO₅P+H]⁺: 602.2096, found: 602.2080.

Compound 5j. White solid; yield 189 mg, 99%; mp 79-80° C.; ¹H NMR (400MHz, CDCl₃) δ=7.76-7.75 (d, J=7.5 Hz, 2H), 7.70-7.63 (m, 6H), 7.60-7.53(m, 4H), 7.48-7.42 (m, 6H), 7.41-7.37 (t, J=7.5 Hz, 2H), 7.31-7.27 (t,J=7.4 Hz, 2H), 5.42-5.37 (m, 1H), 5.23 (s, 2H), 4.41-4.39 (d, J=7.1 Hz,2H), 4.24-4.21 (t, J=7.0 Hz, 1H), 4.06-4.05 (d, J=5.6 Hz, 2H); ¹³C NMR(100 MHz, CDCl3) δ=169.9, 156.4, 143.9, 141.4, 139.3, 132.9, 132.6,132.2, 132.1, 131.8, 128.7, 128.6, 128.1, 128.0, 127.9, 127.2, 125.2,120.1, 67.4, 66.4, 47.2, 42.9; ³¹P NMR (162 MHz, CDCl₃) δ=29.33; HRMS(ESI): m/z calcd for [C₃₆H₃₂NO₅P+H]⁺: 588.1940, found: 588.1925.

Compound 5k. White solid; yield, 99%; mp 99-100° C.; ¹H NMR (400 MHz,CDCl₃) δ=7.77-7.75 (d, J=7.6 Hz, 2H), 7.69-7.64 (m, 6H), 7.56-7.53 (t,J=7.4 Hz, 4H), 7.48-7.44 (m, 4H), 7.41-7.36 (m, 4H), 7.31-7.27 (t, J=7.4Hz, 2H), 6.95-6.93 (d, J=8.4 Hz, 2H), 6.87-6.85 (d, J=8.4 Hz, 2H),5.28-5.26 (d, J=7.9 Hz, 1H), 5.22-5.13 (q, J=8.5 Hz, 2H), 4.70-4.68 (m,1H), 4.44-4.32 (m, 2H), 4.21-4.18 (t, J=6.9 Hz, 1H), 3.09-3.06 (m, 2H),1.30 (s, 9H); ³¹P NMR (100 MHz, CDCl₃) δ=171.5, 155.7, 154.7, 143.9,143.8, 141.4, 139.2, 132.9, 132.6, 132.5, 132.2, 132.1, 129.9, 128.8,128.6, 128.2, 128.0, 127.9, 127.2, 125.2, 124.3, 120.1, 78.6, 67.1,66.5, 55.0, 47.3, 37.8, 28.9; ³¹P NMR (162 MHz, CDCl₃) δ=29.29; HRMS(ESI): m/z calcd for [C₄₇H₄₄NO₆P+H]⁺: 750.2984, found: 750.2966.

Synthesis of compound 6a: Boc-Phe-OBndpp 5a (80 mg) was dissolved in 5mL 60% TFA/DCM and stirred at room temperature. After 1 hour, thesolvent mixture was evacuated, and the crude dissolved in DCM. Afterwashing X2 with 1 M Hel(aq), the organic layer was dried with MgSO₄,filtered, and concentrated to afford crude 6a Hel salt. GAP purificationwas conducted by dissolving the crude in a minimal amount of ethylacetate, followed by precipitation with petroleum ether. The purifiedproduct was isolated via filtration as a white solid; yield 71 mg, 99%;mp 68-71° C. (decomposition); ¹H NMR (400 MHz, CDCl₃) δ=7.57-7.40 (m,12H), 7.10-7.04 (m, 7H), 4.99-4.96 (d, J=10.4 Hz, 2H), 4.41 (bs, 1H),3.43 (bs, 1H), 3.25 (bs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ=169.1, 138.6,134.3, 132.5, 132.3, 132.2, 132.1, 132.0, 131.5, 129.6, 128.8, 128.7,128.6, 128.3, 128.2, 127.5, 67.0, 54.6, 36.6; ³¹P NMR (162 MHz, CDCl₃)δ=29.79; HRMS (ESI): m/z calcd for [C₂₈H₂₆NO₃P+H]⁺: 456.1729, found:456.1725.

Synthesis of HBndpp 7a: Boc-Phe-OBndpp 5a (100 mg) was dissolved in a 5mL mixture of methanol and 10% Pd/C (20 mg). The reaction mixture wasplaced under H₂ atmosphere (balloon) and stirred at room temperature for12 hours. The reaction mixture was then filtered through celite and themethanol evacuated. The crude solid was dissolved in DCM and washed X2with sat. Na₂CO₃(aq) solution. The organic layer was dried over MgSO₄,filtered, and evacuated to afford HBndpp 7a as a white solid; yield, 51mg, 97%; this compound has been previously synthesized via a differentmethod, and NMR data matches that found in the literature³⁰: ¹H NMR (400MHz, CDCl₃) δ=7.68-7.63 (m, 4H), 7.57-7.52 (m, 4H), 7.48-7.44 (m, 4H),7.29-7.26 (m, 2H), 2.41 (s, 3H).

General procedure for Fmoc deprotection and coupling: Fmoc-(AA)n-OBnDppdissolved in 30% Piperidine/DCM (100 mL per gram), and stirred at roomtemperature for 10 minutes. Reaction mixture washed X3 with sat.NH₄Cl(aq), dried over MgSO₄, and filtered. To the resulting DCM solutionwas added 1.2 eq TBTU, 1.2eq Fmoc-AA-OH, and 2.4 eq DIPEA; the couplingreaction was stirred for 20 min. The reaction mixture was then washed X2with sat. NH₄Cl(aq), followed by 0.5 M NaOH X2. The combined organiclayers were dried over MgSO₄, filtered, and evacuated to afford thecrude peptide. GAP purification was performed by dissolving the crudemixture (containing Fmoc-(AA)_(n+1)-OBndpp, NFMP, and tetramethylurea)in a minimal amount of ethyl acetate (with some DCM for longerpeptides), followed by precipitation of the product with petroleumether. The product peptide was removed via vacuum filtration as a whitesolid in quantitative yield.

Compound 9k, Fmoc-Arg(Pbf)-Lys(Boc)-Asp(tBu)-Val-Tyr(tBu)-OBndpp. Whitesolid; yield 3.08 g, 97% (over 3 steps from 6k); mp 124-125° C.;Retention time on analytical NP-HPLC with 0.1% ethanolamine in IPA asthe eluent: 8.85 min, 92.0% purity; HRMS (ESI): m/z calcd for[C₉₀H₁₁₄N₉O₁₇PS+H]⁺: 1657.7903, found: 1657.7871.

Deprotection of side-chain protecting groups:Fmoc-Arg(Pbf)-Lys(Boc)-Asp(tBu)-Val-Tyr(tBu)-OBnDpp 9k was dissolved in100 mL 30% Piperidine/DCM and stirred at room temp for 10 minutes. Thereaction mixture was then washed X2 with saturated NH₄Cl(aq), dried overMgSO₄, filtered and evacuated. The crude was then dissolved inTFA/DCNI/H₂O (6/3/1) and stirred at room temp for 1 hour. The reactionmixture was evacuated to saturation, and then the product peptideprecipitated with diethyl ether. Peptide 10k was obtained afterfiltration as a white solid and directly used for the next step.

Deprotection of BnDpp: To 100 mg dry Pd/C in a hydrogenation bottle wasadded H-RKDVY-OBnDpp 10k in 150 mL methanol. The bottle was placed under70 PSI H₂ atmosphere and shaken at room temperature for 24 hours. Thereaction mixture was filtered through celite, and evacuated to dryness.The crude was dissolved in a mixture of 10% acetic acid (aq) andchloroform, after which the aqueous layer was washed X2 with chloroform.Evacuation of the aqueous layer afforded thymopentin as a white solid;yield, 1.09 g, 87%; Retention time on analytical RP-HPLC with 50% MeCNin 0.06% TFA/H₂O as the eluent: 1.24 min, 98.9% purity; HRMS (ESI): m/zcalcd for [C₃₀H₄₉N₉O₉+H]⁺: 680.3731, found: 680.3730.o our delight, HPLCanalysis of the product peptide reveals that the compound is nearly 99%pure without any column chromatography, recrystallization, or polymersupports. The GAP group can be recovered simply by evacuating thechloroform layer after extraction. Subjecting this raw material to thesynthesis methods in FIG. 2 can regenerate BndppOH.

Further GAP Groups and Attachment Methods

FIG. 6 depicts representative protecting groups that can be used inembodiments of the present invention. FIGS. 7-8 depict alternativeprocesses that can be used to develop BndppOH (alternative of theprocess shown in FIG. 2) and to develop other representative protectinggroups, such as set forth in FIG. 6.

FIGS. 4A-4B each depicts a schematic for the process of attaching theprotecting group of FIG. 2 to various amino acids. Other process forattaching protecting groups are shown in FIGS. 9A-9B and 10A-10B. FIG.9A depicts a schematic for the process of attaching the protecting groupof “BnDppYH” to various amino acids. FIG. 9B depicts the protectinggroup “BnDppYH” utilized in the schematic for the process shown in FIG.9A. FIG. 10A depicts a schematic for the process of attaching theprotecting group of “BzDppOH” to various amino acids. FIG. 10B depictsthe protecting group “BzDppOH” utilized in the schematic for the processshown in FIG. 10A.

These additional protecting groups can be used for peptide synthesis inthe same fashion as “BnDppOH” consistent with embodiments of the presentinvention. The peptide coupling reactions for these additionalprotecting groups can be conducted in ethyl acetate as well asdichloromethane.

Those skilled in the art will recognize that the methods and systems ofthe present disclosure may be implemented in many manners and as suchare not to be limited by the foregoing exemplary embodiments andexamples. In other words, functional elements being performed by singleor multiple components, in various combinations of hardware and softwareor firmware, and individual functions, may be distributed among varioussoftware applications at either the client level or server level orboth. In this regard, any number of the features of the differentembodiments described herein may be combined into single or multipleembodiments, and alternate embodiments having fewer than, or more than,all of the features described herein are possible.

Functionality may also be, in whole or in part, distributed amongmultiple components, in manners now known or to become known. Thus,myriad combinations are possible in achieving the functions, features,and preferences described herein. Moreover, the scope of the presentdisclosure covers conventionally known manners for carrying out thedescribed features as well as those variations and modifications thatmay be made to the processes, composition, or compounds described hereinas would be understood by those skilled in the art now and hereafter.

Furthermore, the embodiments of methods presented and described asdiagrams, schematics or flowcharts in this disclosure (such as theFigures) are provided by way of example in order to provide a morecomplete understanding of the technology. The disclosed methods are notlimited to the operations and logical flow presented herein. Alternativeembodiments are contemplated in which the order of the variousoperations is altered and in which sub-operations described as beingpart of a larger operation are performed independently.

While various embodiments have been described for purposes of thisdisclosure, such embodiments should not be deemed to limit the teachingof this disclosure to those embodiments. Various changes andmodifications may be made to the elements and operations described aboveto obtain a result that remains within the scope of the systems andprocesses described in this disclosure.

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1. A protecting group for Group Assisted Purification (GAP) peptidesynthesis, selected from the group consisting of:

wherein: R is selected from the group consisting of: H, Me, and OMe; Yis selected from the group consisting of: O, S, and NH; and X isselected from the group consisting of: O, S, and NH. 2.-7. (canceled) 8.A method of attaching a protecting group of claim 1 to an ammo acid,wherein the method comprises: reacting a protecting group of claim 1with amino acid compound:


9. The method of claim 8, wherein the method comprises the steps of:

wherein BnDppOH is a protecting group of claim 1; and wherein Pg isselected from the group consisting of: Cbz, Fmoc, Boc, Bn, Fm, and tBu;and wherein Z is a general variable.
 10. The method of claim 9, whereinthe protecting group is compound:


11. The method of claim 8, wherein the method comprises the steps of:

wherein BnDpp YH is a protecting group of claim 1; and wherein Pg isselected from the group consisting of: Cbz, Fmoc, Boc, Bn, Fm, and tBu;and wherein Z is a general variable.
 12. The method of claim 11, whereinthe protecting group is selected from the group consisting of:

wherein R is selected from the group consisting of: H, Me, and OMe; andwherein X is selected from the group consisting of: O, S, and NH. 13.The method of claim 8, wherein the method comprises the steps of:

wherein BnDppYH is a protecting group of claim 1; and wherein Pg isselected from the group consisting of: Cbz, Fmoc, Boc, Bn, Fm, and tBu;and wherein Z is a general variable.
 14. The method of claim 8, whereinthe method comprises the steps of:

wherein BzDppOH is a protecting group of claim 1; and wherein Pg isselected from the group consisting of: Cbz, Fmoc, Boc, Bn, Fm, and tBu;and wherein Z is a general variable.
 15. The method of claim 14, whereinthe protecting group is selected from the group comprising:

wherein R is selected from the group consisting of: H, Me, and OMe; andwherein X is selected from the group consisting of: O, S, and NH
 16. Amethod of performing a Group Assisted Purification (GAP) peptidesynthesis, wherein the method comprises the steps of attaching aprotecting group of claim 1 to an amino acid followed by Fmoc-tBu-basedsolution phase peptide synthesis (SolPPS) coupling reactions on theresulting amino acid having the attached protecting group.
 17. Themethod of claim 16, wherein the reaction occurs in ethyl acetate. 18.The method of claim 16, wherein the reaction occurs in dichloromethane.